Project Summary/Abstract Research: Tetraploid cells, which are a common byproduct of cell division failures, are genetically unstable and have the capacity to facilitate tumorigenesis. Consequently, cells possess p53-dependent tumor suppression mechanisms that limit the further proliferation of these cells by promoting a durable G1 cell cycle arrest. However, unlike other common cellular insults that activate p53 and promote G1 arrest, such as the DNA damage response, the mechanisms governing G1 arrest in response to tetraploidy remain largely unexplored. A major goal of this proposal is to uncover how cells 'sense' cellular changes associated with tetraploidy and then relay that information into the p53 pathway to prevent cell cycle progression. To address this fundamental question, I have developed a novel genome-wide RNAi screening assay to comprehensively identify proteins that are necessary to activate or maintain G1 cell cycle arrest after cytokinesis failure and the induction of tetraploidy. Results from this screen, in combination with complementary bioinformatic and biochemical approaches, will illuminate the nature of the stresses associated with tetraploidy: this includes identifying how these stresses are sensed, how they feed into signaling cascades that activate p53, and how they are ultimately overcome by cancer cells. Strong gene hits from this screen will be examined to determine if they are similarly required to activate G1 arrest in response to other common cellular defects, and whether they are commonly lost or mutated in human cancers. Ultimately, the most interesting candidates genes will be tested for tumor suppressive activities in vitro and in vivo. Overall, the aims described in this proposal have strong potential to identify the cellular defects associated with tetraploidy, as well as to uncover novel regulatory mechanisms of p53. Moreover, this work may also lead to the identification of new pathways that can be targeted by chemotherapeutics to reinforce G1 arrest in abnormal cancer cells. Candidate Career Goals: My long-term career goal is to obtain a tenure-track faculty position at a leading academic institution and establish a lab that is at the forefront of unraveling the many mysteries of cell cycle progression. In particular, I have noticed a great divide in the approaches used by cell biologists and cancer biologists to understand both the causes and consequences of cancer: in my lab, I plan on bridging this gap by combining my pre-existing cell biological expertise with the new training opportunities in genome-wide screening, bioinformatics, and transformation assays described in this proposal. Ultimately, my aim is to blend these two fields in order to take a unique multifaceted experimental approach to probe the detailed mechanisms governing cell cycle progression. The K99/R00 award will provide the protected time I need for focused advanced training in order to achieve this career goal. I expect the mentored phase of this proposal, which includes completing the genome-wide screen and characterizing novel pathways that regulate p53, to take 1-2 years and result in at least one high quality publication. The following independent phase of the award will then permit me to further explore the activation and regulation of these signaling pathways, as well as to uncover new tumor suppressor pathways. Together, these data will be used to justify future studies proposed in an RO1 grant application that I expect to submit at the beginning of the third year of the independent phase. Environment: The Department of Pediatric Oncology at the Dana-Farber Cancer Institute (DFCI) and Harvard Medial School has an internationally recognized research program that houses a number of expert researchers in the areas of cell proliferation, tumorigenesis, and cancer cell biology. I have assembled a stellar mentoring and advisory committee (Dr. David Pellman, Dr. William Kaelin, Dr. Ron Depinho, Dr. Matt Meyerson, and Dr. William Hahn) that will greatly benefit my research and training experience. I will meet with members of this committee informally as needed for specific scientific or experimental advice, and formally twice a year to discuss the general progress of my project, identify key future directions, and plan for my transition to independence. In addition, the Harvard Medical Area has all of the necessary physical resources required to for me to complete the proposed training and research studies, including facilities for FACS sorting, mass spectrometry, microarray analysis, and genome-wide screening.